Gül Erdemli

A New Perspective in the Field of Cardiac Safety Testing through the Comprehensive In Vitro Proarrhythmia Assay Paradigm

For the past decade, cardiac safety screening to evaluate the propensity of drugs to produce QT interval prolongation and Torsades de Pointes (TdP) arrhythmia has been conducted according to ICH S7B and ICH E14 guidelines. Central to the existing approach are hERG channel assays and in vivo QT measurements. Although effective, the present paradigm carries a risk of unnecessary compound attrition and high cost, especially when considering costly thorough QT (TQT) studies conducted later in drug development. The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative is a public-private collaboration with the aim of updating the existing cardiac safety testing paradigm to better evaluate arrhythmia risk and remove the need for TQT studies. It is hoped that CiPA will produce a standardized ion channel assay approach, incorporating defined tests against major cardiac ion channels, the results of which then inform evaluation of proarrhythmic actions in silico, using human ventricular action potential reconstructions. Results are then to be confirmed using human (stem cell–derived) cardiomyocytes. This perspective article reviews the rationale, progress of, and challenges for the CiPA initiative, if this new paradigm is to replace existing practice and, in time, lead to improved and widely accepted cardiac safety testing guidelines.

Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation.

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.

Cardiac Safety Implications of hNav1.5 Blockade and a Framework for Pre-Clinical Evaluation

The human cardiac sodium channel (hNav1.5, encoded by the SCN5A gene) is critical for action potential generation and propagation in the heart. Drug-induced sodium channel inhibition decreases the rate of cardiomyocyte depolarization and consequently conduction velocity and can have serious implications for cardiac safety. Genetic mutations in hNav1.5 have also been linked to a number of cardiac diseases. Therefore, off-target hNav1.5 inhibition may be considered a risk marker for a drug candidate. Given the potential safety implications for patients and the costs of late stage drug development, detection, and mitigation of hNav1.5 liabilities early in drug discovery and development becomes important. In this review, we describe a pre-clinical strategy to identify hNav1.5 liabilities that incorporates in vitro, in vivo, and in silico techniques and the application of this information in the integrated risk assessment at different stages of drug discovery and development.

IonFlux: A Microfluidic Patch Clamp System Evaluated with Human Ether-a`-go-go Related Gene Channel Physiology and Pharmacology

Ion channel assays are essential in drug discovery, not only for identifying promising new clinical compounds, but also for minimizing the likelihood of potential side effects. Both applications demand optimized throughput, cost, and predictive accuracy of measured membrane current changes evoked or modulated by drug candidates. Several competing electrophysiological technologies are available to address this demand, but important gaps remain. We describe the industrial application of a novel microfluidic-based technology that combines compounds, cells, and buffers on a single, standard well plate. Cell trapping, whole cell, and compound perfusion are accomplished in interconnecting microfluidic channels that are coupled to pneumatic valves, which emancipate the system from robotics, fluidic tubing, and associated maintenance. IonFlux™ is a state-of-the-art, compact system with temperature control and continuous voltage clamp for potential application in screening for voltage- and ligand-gated ion channel modulators. Here, ensemble recordings of the IonFlux system were validated with the human Ether-à-go-go related gene (hERG) channel (stably expressed in a Chinese hamster ovary cell line), which has established biophysical and pharmacological characteristics in other automated planar patch systems. We characterized the temperature dependence of channel activation and its reversal potential. Concentration response characteristics of known hERG blockers and control compounds obtained with the IonFlux system correlated with literature and internal data obtained on this cell line with the QPatch HT system. Based on the biophysical and pharmacological data, we conclude that the IonFlux system offers a novel, versatile, automated profiling, and screening system for ion channel targets with the benefit of temperature control.

The translocation kinetics of antibiotics through porin OmpC: Insights from structure-based solvation mapping using WaterMap

Poor permeability of the lipopolysaccharide‐based outer membrane of Gram‐negative bacteria is compensated by the existence of protein channels (porins) that selectively admit low molecular weight substrates, including many antibiotics. Improved understanding of the translocation mechanisms of porin substrates could help guide the design of antibiotics capable of achieving high intracellular exposure. Energy barriers to channel entry and exit govern antibiotic fluxes through porins. We have previously reported a hypothesis that the costs of transferring protein solvation to and from bulk medium underlie the barriers to protein‐ligand association and dissociation, respectively, concomitant with the gain and loss of protein‐ligand interactions during those processes. We have now applied this hypothesis to explain the published rates of entry (association) and exit (dissociation) of six antibiotics to/from reconstituted E. coli porin OmpC. WaterMap was used to estimate the total water transfer energies resulting from transient occupation by each antibiotic. Our results suggest that solvation within the porin cavity is highly energetically favorable, and the observed moderately fast entry rates of the antibiotics are consistent with replacement of protein‐water H‐bonds. The observed ultrafast exit kinetics is consistent with the lack of intrachannel solvation sites that convey unfavorable resolvation during antibiotic dissociation. These results are aligned with known general relationships between antibiotic efficacy and physicochemical properties, namely unusually low logP, reflecting an abundance of H‐bond partners. We conclude that antibiotics figuratively “melt” their way through porin solvation at a rate determined by the cost of exchanging protein‐solvent for protein‐antibiotic H‐bonds. Proteins 2013.

Cardiac Ion Channel Safety Profiling on the IonWorks Quattro Automated Patch Clamp System

The normal electrophysiologic behavior of the heart is determined by the integrated activity of specific cardiac ionic currents. Mutations in genes encoding the molecular components of individual cardiac ion currents have been shown to result in multiple cardiac arrhythmia syndromes. Presently, 12 genes associated with inherited long QT syndrome (LQTS) have been identified, and the most common mutations are in the hKCNQ1 (LQT1, Jervell and Lange-Nielson syndrome), hKCNH2 (LQT2), and hSCN5A (LQT3, Brugada syndrome) genes. Several drugs have been withdrawn from the market or received black box labeling due to clinical cases of QT interval prolongation, ventricular arrhythmias, and sudden death. Other drugs have been denied regulatory approval owing to their potential for QT interval prolongation. Further, off-target activity of drugs on cardiac ion channels has been shown to be associated with increased mortality in patients with underlying cardiovascular diseases. Since clinical arrhythmia risk is a major cause for compound termination, preclinical profiling for off-target cardiac ion channel interactions early in the drug discovery process has become common practice in the pharmaceutical industry. In the present study, we report assay development for three cardiac ion channels (hKCNQ1/minK, hCa(v)1.2, and hNa(v)1.5) on the IonWorks Quattro™ system. We demonstrate that these assays can be used as reliable pharmacological profiling tools for cardiac ion channel inhibition to assess compounds for cardiac liability during drug discovery.

Phenotypic screening in the 21st century

Since the advent of molecular cloning, target based screening has become the norm in pharmaceutical drug discovery. A large number of potential drug targets have been cloned and functionally expressed, and enormous progress has been made in the development, miniaturization and automation of cell based assays on target molecules recombinantly expressed in mammalian cell lines. This approach has delivered many clinical candidates but relatively few new drugs. Target based screening is likely to provide very good drug candidates for monogenic diseases, and the following collection of manuscripts is not meant to discourage the use of target based approaches. However, most of the more prevalent human diseases are most likely multifactorial and require interaction with multiple targets to produce clinically meaningful efficacy. In addition, high potency, selective interaction with a single target may increases the risk of adverse events or be limited by redundancies and adaptive resistance. Here, target agnostic approaches using phenotypic assays may offer significant benefit. Making such approaches viable requires addressing a number of challenges. This e-book attempts to discuss some of these challenges and illustrate recent advances.

Implications of Dynamic Occupancy, Binding Kinetics, and Channel Gating Kinetics for hERG Blocker Safety Assessment and Mitigation

Blockade of the hERG potassium channel prolongs the ventricular action potential (AP) and QT interval, and triggers early after depolarizations (EADs) and torsade de pointes (TdP) arrhythmia. Opinions differ as to the causal relationship between hERG blockade and TdP, the relative weighting of other contributing factors, definitive metrics of preclinical proarrhythmicity, and the true safety margin in humans. Here, we have used in silico techniques to characterize the effects of channel gating and binding kinetics on hERG occupancy, and of blockade on the human ventricular AP. Gating effects differ for compounds that are sterically compatible with closed channels (becoming trapped in deactivated channels) versus those that are incompatible with the closed/closing state, and expelled during deactivation. Occupancies of trappable blockers build to equilibrium levels, whereas those of non-trappable blockers build and decay during each AP cycle. Occupancies of ~83% (non-trappable) versus ~63% (trappable) of open/inactive channels caused EADs in our AP simulations. Overall, we conclude that hERG occupancy at therapeutic exposure levels may be tolerated for nontrappable, but not trappable blockers capable of building to the proarrhythmic occupancy level. Furthermore, the widely used Redfern safety index may be biased toward trappable blockers, overestimating the exposure-IC50 separation in nontrappable cases.

Structure‐kinetic relationship of carbapenem antibacterials permeating through E. coli OmpC porin

The emergence of Gram‐negative “superbugs” exhibiting resistance to known antibacterials poses a major public health concern. Low molecular weight Gram‐negative antibacterials are believed to penetrate the outer bacterial membrane (OM) through porin channels. Therefore, intracellular exposure needed to drive antibacterial target occupancy should depend critically on the translocation rates through these proteins and avoidance of efflux pumps. We used electrophysiology to study the structure‐translocation kinetics relationships of a set of carbapenem antibacterials through purified porin OmpC reconstituted in phospholipid bilayers. We also studied the relative susceptibility of OmpC+ and OmpC‐ E. coli to these compounds as an orthogonal test of translocation. Carbapenems exhibit good efficacy in OmpC‐expressing E. coli cells compared with other known antibacterials. Ertapenem, which contains an additional acidic group compared to other analogs, exhibits the fastest entry into OmpC (kon ≈ 2 × 104 M−1 s−1). Zwitterionic compounds with highly polar groups attached to the penem‐2 ring, including panipenem, imipenem and doripenem exhibit faster kon (>104 M−1 s−1), while meropenem and biapenem with fewer exposed polar groups exhibit slower kon (∼5 × 103 M−1 s−1). Tebipenem pivoxil and razupenem exhibit ∼13‐fold slower kon (∼1.5 × 103 M−1 s−1) than ertapenem. Overall, our results suggest that (a) OmpC serves as an important route of entry of these antibacterials into E. coli cells; and (b) that the structure‐kinetic relationships of carbapenem translocation are governed by H‐bond acceptor/donor composition (in accordance with our previous findings that the enthalpic cost of transferring water from the constriction zone to bulk solvent increases in the presence of exposed nonpolar groups). Proteins 2014; 82:2998–3012.

Assay Development and Validation for the Muscle Nicotinic Receptor Using the Qpatch HTX™ Automated Patch Clamp System

Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research Inc., 250 Massachusetts Avenue Cambridge, MA 02139, USA.QPatch HTX is an automated patch clamp system with greatly increased throughput over conventional patch clamp methods. In this study, we evaluated the suitability of this system for the characterization of the fast desensitizing ligand-gated ion channel coupled with alpha1 nicotinic acetylcholine receptors (α1nAChR). HEK293 cells stably expressing human α1nAChR were obtained from Millipore. All experiments were carried out with the QPatch HTX, which performs 48 parallel and independent patch-clamp recordings on a disposable QPlate X with 10 holes per measurement site. Brief applications of acetylcholine (ACh) produced fast inward currents characteristic of the α1nAChR recorded by conventional patch clamp in heterologous expression systems and the native channel. The EC50 values for ACh ranged from 3 to 10 µM (n=10). Nicotine and epibatidine also elicited inward currents with EC50 values of 110 µM and 4 µM, respectively (n=6 & 12). The ACh-induced current was resistant to bath applications of atropine up to 1 µM and muscarine did not elicit a response, showing that the ACh-induced inward current is mediated by nAChR. Non expressing HEK293 cells did not respond to ACh. The ACh-mediated currents were blocked by α-bungarotoxin and pancuronium in a dose dependent manner (IC50= 80 and 0.8 nM, respectively).In conclusion, this study demonstrated that fast desensitizing ligand-gated ion channel can be efficiently targeted with QPatch HTX system for characterization of physiological and pharmacological properties.